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Association of _s-Subunit of the G_s Protein with Microfilaments and Microtubules: Implication during Adrenocorticotropin Stimulation in Rat Adrenal Glomerulosa Cells¹

Service of Endocrinology, Departments of Medicine (M.C., N.G.-P.), Anatomy and Cell Biology (M.C., N.G.-P.), and Physiology and Biophysics (M.D.P.), Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4

Address all correspondence and requests for reprints to: Dr. Nicole Gallo-Payet, Service of Endocrinology, Department of Medicine, Faculty of Medicine, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, Quebec, Canada J1H 5N4. E-mail: n.gallo{at}courrier.usherb.ca

	Abstract

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The aim of the present study was to investigate if and how microfilaments and microtubules could be involved in the early events of ACTH action. In primary cultures of rat glomerulosa cells, a 30-min preincubation with either 10 µM colchicine (a microtubule-disrupting agent) or 10 µM cytochalasin B (a microfilament-disrupting agent) decreased ACTH-induced cAMP production. Moreover, colchicine decreased cAMP production induced by fluoroaluminate (a nonspecific activator of all G proteins), but not of forskolin (which directly activates adenylyl cyclase). These results indicate that microtubules appear to be essential for the G_s protein activation. In contrast, cytochalasin B decreased the stimulating effect of both fluoroaluminate and forskolin, indicating that microfilaments may be involved in both G_s and adenylyl cyclase activations. Analyses of microfilament- and microtubule-enriched fractions and immunoprecipitation of actin and tubulin indicated that the _s-subunit of the G_s protein was associated with both structures. Stimulation of cells with ACTH induced a rapid increase (within 1 min) in the levels of microfilaments, microtubules, and _s associated with the membrane. In addition, ACTH stimulation of cAMP production was very sensitive to Ca²⁺, without any stimulation in Ca²⁺-free medium. Under these conditions, actin filaments were short and formed a dense network. These observations suggest that the Ca²⁺-free medium stabilized the actin fibers in such a way that activation by ACTH failed, further documenting the importance of microfilaments in cAMP production.

	Introduction

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SEVERAL STUDIES have demonstrated that the cytoskeleton plays an important role in adrenal steroidogenesis. Cytochalasin B, a disrupter of microfilament organization, decreases the basal release of steroids and inhibits all stimulus-induced adrenal corticosteroidogenesis in rat, frog, and human (1, 2, 3). Colchicine and vinblastine (two microtubule-disrupting agents) and ß-ß'-iminodipropionitrile, an intermediate filament inhibitor, do not affect the basal release of steroids, but respectively decrease the effects of ACTH (4) and angiotensin II (5). Microfilaments are clearly involved in the transport of cholesterol from lipid droplets to endoplasmic reticulum and mitochondria, two organelles involved in steroidogenesis, whereas microtubules may act earlier during the process of ACTH stimulation (1, 6). Indeed, vinblastine decreases ACTH-stimulated corticosterone secretion, but not (Bu)₂cAMP-induced stimulation, whereas cytochalasin B causes a reduction of cAMP-induced stimulation, suggesting that microtubules may be involved in the transduction process that triggers cAMP production (3, 7). In contrast, Ray and Strott (8) and Saltarelli et al. (9) in adrenal cells and Aharoni et al. (10) in granulosa cells found that cytoskeletal disruption is associated with an increase in cAMP production and steroid secretion. All of these effects have been recently summarized by Feuillolley and Vaudry (11). The morphological changes in the microtubule and microfilament networks during ACTH stimulation have been extensively studied in the mouse adrenocortical tumor cell line Y-1 (12). In addition, several studies suggest that the cytoskeleton may be involved in signal transduction processes. Receptors (13, 14, 15), G proteins (16, 17, 18), and adenylyl cyclase (19, 20, 21) have been found to be associated with both microfilaments and microtubules.

In the rat adrenal gland, there is little information on the direct role of the cytoskeleton in the early events of ACTH action, namely cAMP production, and on a possible association of G_s with cytoskeleton. The aims of the present study were 1) to study the influence of cytoskeletal disruption on cAMP production induced by ACTH, 2) to analyze the time-dependent changes in microfilament and microtubule organization, and 3) to investigate whether the _s-subunit of the G_s protein could be associated with microfilaments and microtubules.

	Materials and Methods

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Chemicals
The chemicals used in the present study were obtained from the following sources: [³H]adenine (24 Ci/mmol) from Amersham (Oakville, Canada); aldosterone antiserum from ICN Biochemicals (Costa Mesa, CA); [³H]aldosterone (72 Ci/mmol) from New England Nuclear (Boston, MA); cytochalasin B, colchicine, ATP, cAMP, taxol, and deoxyribonuclease from Sigma Chemical Co. (St. Louis, MO); ACTH-(1–24) peptide (Cortrosyn) from Organon (Toronto, Canada); collagenase, MEM (Eagle’s medium), and OPTI-MEM medium from Life Technologies (Burlington, Canada); anti-ß-tubulin monoclonal antibody from Sigma or Amersham; taxol and actin antibody from Boehringer Mannheim (clone C4, Montreal, Canada); anti-_s from New England Nuclear-DuPont (Mississauga, Canada); anti-_q from Dr. Gilles Guillon (INSERM U-401, Monpellier, France); anti-IgG antibody from Calbiochem (La Jolla, CA); rhodamine-phalloidin from Molecular Probes (Eugene, OR); polyvinylidene difluoride membranes and Immobilon P from Millipore (Bedford, CA); and Vectashield from Vector Laboratories (Burlingame, CA). All other chemicals were of A grade purity.

Preparation of glomerulosa cells
The zonae glomerulosa were obtained from adrenal glands of female Long-Evans rats, weighing 200–250 g, and were isolated according to the method described in detail previously (22, 23). The successive steps of zona glomerulosa isolation and cell dissociation were performed in MEM (supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin). After a 20-min incubation at 37 C in collagenase (2 mg/ml, 4 capsules/ml) and deoxyribonuclease (25 µg/ml), the cells were disrupted by gentle aspiration with a sterile 10-ml pipette, filtered, and centrifuged for 10 min at 100 x g. They were then resuspended in OPTI-MEM medium supplemented with 2% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin and plated in 35-mm tissue culture dishes (for cAMP experiments) or 16-mm multiwell plates (for steroid measurements) at a density of approximately 1 x 10⁵ cells/multiwell or dish, respectively. The cells were cultured at 37 C in a humidified atmosphere of 95% air-5% CO₂. The culture medium was changed every day, and the cells were used after 3 days of culture. At this time, cell density was approximately 1–3.0 x 10⁵ cells/dish or well in a multiwell plate.

Incubations for measurement of aldosterone secretion
Before each experiment, the medium of cultured cells was aspirated, and the cells were washed twice with cold Hanks’ buffered saline (HBS; 130 mM NaCl, 3.5 mM KCl, 1.8 mM CaCl₂, 0.5 mM MgCl₂, 2.5 mM NaHCO₃, and 5 mM HEPES) supplemented with 1 g/liter glucose and 0.5% BSA. The cells were incubated in 1 ml, consisting of 0.9 ml HBS-glucose supplemented with 0.5% BSA-0.1 mg/ml bacitracin and 0.1 ml of stimuli. After a 2-h incubation at 37 C in an atmosphere of 95% air-5% CO₂, the incubation medium was removed by aspiration and stored at -20 C until assayed aldosterone in the medium was determined by RIA, using specific antisera and tritiated steroid as tracer.

cAMP determination
Intracellular cAMP production was determined by measuring the conversion of [³H]ATP to [³H]cAMP, as previously described (24). In short, cultured cells were incubated at 37 C in OPTI-MEM culture medium containing 2 µCi/ml [³H]adenine. After 1 h, the cultures were washed with HBS buffer and incubated in the same buffer containing 1 mM isobutylmethylxanthine for 15 min at 37 C. The hormones or drugs were then added to the incubation medium for an additional 15 min at 37 C. The reaction was ended by aspiration of the medium. Cells were scraped with a rubber policeman, and 100 µl of a cold solution of ATP and cAMP (5 mM each) were added to the mixture. Cellular membranes were pelleted at 5000 x g for 15 min, and the supernatants were sequentially chromatographed on Dowex and alumina columns according to the method of Salomon et al. (25), allowing the separation of [³H]ATP nucleotide (primarily [³H]adenine) from [³H]cAMP. cAMP formation was expressed as: % conversion = ([³H]cAMP/[³H]cAMP + [³H]ATP) x 100/15 min.

Membrane preparation
After hormonal stimulation, 3-day cultured cells were washed twice with HBS buffer and then with 10 mM ice-cold Tris-HCl buffer (containing 0.5 mM EDTA, 1 mM MgCl₂, 28 mM phenylmethylsulfonylfluoride, 0.04 U/ml aprotinin, and 1 mM benzamidine, pH 8.0). The cells were then scraped from the substratum with a rubber policeman and homogenized in a sonicator. Cell extracts were centrifuged at 700 x g, and the resultant supernatant was centrifuged at 30,000 x g to obtain the membrane fraction. The membrane fraction was resuspended in 50 mM Tris-HCl buffer (containing 2 mM EDTA, 5 mM MgCl₂, and 250 mM sucrose) and stored at -20 C for subsequent Western blot assays.

Preparation of microtubules
Preparations enriched in microtubules were extracted from cells grown in 60-mm petri dishes as described by Solomon (26) with some modifications. The cells were pretreated with 1 mM taxol for 2 h before extraction of microtubules. At this concentration, taxol stabilizes microtubules without promoting polymerization. The culture medium was then aspirated and replaced with PM2G buffer (0.1 M PIPES, 2 M glycerol, 5 mM MgCl₂, 2 mM EGTA, 0.04 TIU/ml aprotinin, 2 mM phenylmethylsulfonylfluoride, and 1 mM benzamidine, pH 6.9) containing taxol (1 mM). Cells were scraped from the substratum with a rubber policeman and transferred to a 15-ml conical tube and centrifuged at 1,000 x g for 5 min at 37 C. The cell pellet was then extracted with PM2G buffer containing 1% Nonidet P-40 and 1 mM taxol. After a 15-min incubation at 37 C, the suspension was centrifuged at 1,000 x g for 5 min at 37 C. The pellet containing the microtubules was then solubilized in 125 mM Tris buffer, pH 6.8, containing 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol and heated to 100 C for 5 min. After centrifugation at 10,000 x g for 5 min, the supernatant was stored at -20 C for subsequent Western blot analysis. For total cell extracts, cells were grown in 60-mm petri dishes, washed twice with HBS buffer, and solubilized as described above.

Extraction of microfilaments
Enriched microfilament preparations were extracted from cells grown in 60-mm petri dishes as described by Phillips et al. (27). Culture medium was aspirated and changed for HBS buffer. Cells were scraped from the substratum with a rubber policeman and transferred in a 15-ml conical tube. Cells were centrifuged at 100 x g for 5 min at room temperature. One hundred milliliters of Triton solution (1% Triton X-100, 10 mM EGTA, and 0.1 M Tris-HCl, pH 7.4) was added, and the solution was transferred to 1.5-ml microcentrifuge tubes. After a 10-min incubation at 0 C, the preparation was centrifuged at 8000 x g for 4 min at room temperature. Triton-soluble G-actin fraction was contained in the supernatant. The pellet, which corresponds to the Triton-insoluble fraction, was solubilized in 2% SDS-2% 2-mercaptoethanol (vol/vol). After a 10-min incubation at 100 C, F-actin was solubilized. Both fractions were aliquoted and frozen for subsequent Western blot analysis.

Western blotting
Samples from an equivalent number of cells were compared in each experiment. Samples were separated on 4–15% SDS-polyacrylamide gels. Proteins were transferred electrophoretically to polyvinylidene difluoride membranes. Membranes were blocked with 1% gelatin and 0.05% Tween-20 in Tris-buffered saline (TBS; pH 7.5). After three washes with TBS-Tween 20 (0.05%), membranes were incubated with anti-ß tubulin (dilution, 1:250), anti-actin (dilution, 1:100), or anti-_s (dilution, 1:1000) for 3 h at room temperature, followed by four washes with TBS-Tween-20. Detection was accomplished using horseradish peroxidase-conjugated antimouse antibody for actin and tubulin (Amersham) or antirabbit for _s and the enhanced chemiluminescence detection system (Amersham). The immunoreactive bands were scanned by laser densitometry and expressed in arbitrary units. Note that the two isoforms of _s proteins were analyzed together.

Immunoprecipitation
Glomerulosa cells in 60-mm petri dishes were washed once and stimulated with ACTH (100 nM) as indicated at 37 C. The cells were then washed twice with ice-cold HBS buffer and lysed in TSA buffer [0.1 M Tris-HCl (pH 8.0), 0.14 M NaCl, 0.025% NaN₃, 1% Nonidet P-40, 1% BSA, 1 mM phenylmethylsulfonylfluoride, 1 mM iodoacetamide, 0.2 U/ml aprotinin, and 1 mM benzamidine] for 60 min at 4 C. Lysates were clarified with protein A-Sepharose for 2 h at 22 C, followed by centrifugation at 200 x g for 1 min. For immunoprecipitation of actin or tubulin, the lysates were incubated for 2 h with 2 mg/ml monoclonal antibodies at 22 C. Protein A-Sepharose was added, and incubation was performed overnight at 4 C. Immunocomplexes were washed five times before electrophoresis on 4–15% SDS-polyacrylamide gels and analysis by immunoblotting.

Immunofluorescence
For immunofluorescence studies, cells were plated on plastic coverslips (Starsted, St. Laurent, Canada), grown for 3 days, and treated with appropriate stimuli. For microfilament visualization, cells were fixed for 1 min with 3% (vol/vol) formaldehyde in PBS buffer, permeabilized by incubation for 10 min in PBS-0.1% Triton X-100, and incubated for 20 min at room temperature with 1 U rhodamine/phalloidin solution. For microtubules, _s, and _q detection, cells were fixed for 1 min with 3% (vol/vol) formaldehyde in 80 mM PIPES (pH 6.5), 5 mM EDTA, and 2 mM MgCl₂ and fixed for an additional 8 min with 3% (vol/vol) formaldehyde in 100 mM sodium borate (pH 11) (18). Cells were then incubated for 30 min in PBS-0.1% (vol/wt) sodium borohydride; permeabilized by incubation in PBS-0.2% Triton X-100; incubated overnight at 4 C with anti-ß tubulin (1:50), anti-_s (1:50), or anti-_q (1:50); washed; and further incubated for 60 min at 37 C with a secondary conjugated anti-IgG antibody coupled with fluorescein isothiocyanate (FITC). For double immunofluorescence, cells were fixed and permeabilized as described for microtubule and G protein detections and processed successively with anti-_s or anti-_q and anti-ß tubulin or rhodamine/phalloidin as described above. After washings, cells were postfixed for 20 min with 3% formaldehyde-PBS and incubated in the presence of 50 mM NH₄Cl for 10 min. The coverslips were then mounted in Vectashield mounting medium and examined on a Nikon DM 400 microscope equipped for epifluorescence. B-1E FITC and G-2A rhodamine filters (Nikon, Melville, NY) were used to visualize images.

Electron microscopy studies
The Triton X-100-insoluble preparations were prepared and fixed with 2.5% glutaraldehyde, postfixed with 2% osmium, dehydrated, and embedded in Epon 812. Gold to silver-gray sections were then stained with uranyl acetate and lead citrate and examined in a Phillips 300 electron microscope (Phillips, Mahway, NJ).

Data analysis
The data are presented as the mean ± SE. Statistical analyses of the data were performed using one-way ANOVA. Homogeneity of variance was assessed by Bartlett’s test, and P values were obtained from Dunnett’s tables. n indicates the number of experiments; each was performed in triplicate.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of cytoskeletal disruption on cAMP production induced by ACTH
A 30-min preincubation with colchicine (a microtubule-disrupting agent) did not affect basal cAMP levels, but induced a dose-dependent inhibition of ACTH-induced cAMP production. cAMP production increased from 0.043 ± 0.004 in control cells to 0.55 ± 0.03 (n = 4) in ACTH-stimulated cells (13-fold increase). This stimulation decreased to 0.24 ± 0.03 after colchicine treatment (55.2 ± 6.0% decrease; n = 4; P < 0.001 at 10 µM; Fig. 1A). To determine how microtubules may be involved in the activation of adenylyl cyclase, we measured the dose-dependent effect of colchicine on AlF^-₄- and forskolin-stimulated cAMP production. AlF^-₄, a nonspecific activator of all heterotrimeric G proteins (28), and forskolin, which directly activates the catalytic subunit of adenylyl cyclase (29), stimulated cAMP production by 16- and 50-fold, respectively (n = 4). Results from Fig. 1B show that colchicine induced a dose-dependent inhibition of cAMP production induced by AlF^-₄ (60.2 ± 1.8% decrease; n = 4; P < 0.001 at 10 µM), but did not affect the large increase produced by forskolin.

View larger version (17K): [in this window] [in a new window]   Figure 1. Dose-dependent effect of colchicine on ACTH-stimulated cAMP production in rat glomerulosa cells. Three-day cultures of rat glomerulosa (3 x 105 cells/dish) were labeled with [3H]adenine as described in Materials and Methods. Cells were preincubated for 30 min in the absence or presence of increasing concentrations of colchicine and were further incubated for 15 min at 37 C in HBS medium in the absence () or presence (•) of 100 nM ACTH (A) or in the presence of 30 mM AlF-4 () or 10 µM forskolin (; B). The [3H]cAMP concentrations that accumulated were determined and expressed as a percentage of the total intracellular [3H]ATP, calculated as described in Materials and Methods. Results are the mean ± SE of triplicate determinations in one experiment that is representative of three. When no error bars are shown, they are contained within the symbols.

View larger version (14K): [in this window] [in a new window]   Figure 2. Dose-dependent effect of cytochalasin B on ACTH-stimulated cAMP production in rat glomerulosa cells. Three-day cultures of rat glomerulosa (3 x 105 cells/dish) were labeled with [3H]adenine as described in Materials and Methods. Cells were preincubated for 30 min in the absence or presence of increasing concentrations of cytochalasin B and were further incubated for 15 min at 37 C in HBS medium in the absence () or presence of 100 nM ACTH (•; A), 30 mM AlF-4 (; B), or 10 µM forskolin (; C). The [3H]cAMP concentrations that accumulated were determined and expressed as a percentage of the total intracellular [3H]ATP calculated as described in Materials and Methods. Results are the mean ± SE of triplicate determinations in one experiment that is representative of three. When no error bars are shown, they are contained within the symbols.

View larger version (148K): [in this window] [in a new window]   Figure 3. Effect of ACTH on immunofluorescence labeling of actin in rat glomerulosa cells. Rat glomerulosa cells were cultured for 3 days on plastic coverslips and then incubated for various periods (B, 1 min; C, 5 min; D, 15 min; E, 30 min; F, 2 h) in HBS medium in the absence (A) or presence of 100 nM ACTH (B–F). After formaldehyde fixation and permeabilization with 0.1% Triton X-100, cells were processed for immunofluorescence labeling using rhodamine-phalloidin as described in Materials and Methods. All panels are shown at the same magnification of x3120. Images are representative illustrations of more than 50 cells originating from 3 different experiments.

View larger version (130K): [in this window] [in a new window]   Figure 4. Effect of ACTH on immunofluorescence labeling of ß-tubulin in rat glomerulosa cells. Rat glomerulosa cells were cultured for 3 days on plastic coverslips and then incubated for various periods (B, 1 min; C, 15 min; D, 2 h) in HBS medium in the absence (A) or presence of 100 nM ACTH (B–D). After formaldehyde fixation, PIPES treatment, and permeabilization with 0.2% Triton X-100, cells were processed for immunofluorescence labeling using anti-ß-tubulin antibody and FITC as described in Materials and Methods. All panels are shown at the same magnification of x3120. Images are representative illustration of more than 50 cells originating from 3 different experiments.

Analysis of the levels of tubulin, actin, and _s protein.

View larger version (56K): [in this window] [in a new window]   Figure 5. Western blot analysis of the effect of ACTH on the levels of tubulin and actin in rat glomerulosa cells. Three-day cultures of rat glomerulosa were incubated at 37 C in HBS medium in the absence (lane 1) or presence of 100 nM ACTH for 1 min (lane 2), 15 min (lane 3), or 2 h (lane 4). Membrane fractions (A and D), Triton-insoluble fraction (B), cell homogenate (C and F), and microtubule preparation (E) were immunoblotted for actin (A–C) and ß-tubulin (D–F). Cytoskeletal fractions from equivalent number of cells were analyzed in parallel. Cytoskeletal proteins were detected by chemiluminescence as described in Materials and Methods. Numbers on the right indicate the positions of molecular mass markers (kilodaltons). Blots are representative illustrations of results obtained in three independent experiments.

View larger version (70K): [in this window] [in a new window]   Figure 6. Western blot analysis of the effect of ACTH on the level of s-subunit of Gs protein. Three-day cultures of rat glomerulosa were incubated at 37 C in HBS medium in the absence (lane 1) or presence of 100 nM ACTH for 1 min (lane 2), 15 min (lane 3), or 2 h (lane 4). Membrane fractions (A), Triton-insoluble fraction (B), and microtubule preparation (C) were immunobloted for s protein determination. Cytoskeletal fractions from equivalent numbers of cells were analyzed in parallel. Cytoskeletal proteins were detected by chemiluminescence as described in Materials and Methods. Numbers on the right indicate the positions of molecular mass markers (kilodaltons). Blots are representative illustrations of the results obtained in three independent experiments.

View larger version (37K): [in this window] [in a new window]   Figure 7. Immunoprecipitation analysis of the effect of ACTH on the level of s associated with actin and tubulin. Three-day cultures of rat glomerulosa were incubated at 37 C in HBS medium in the absence (lane 1) or presence of 100 nM ACTH for 15 min (lane 2). Cell lysates were immunoprecipitated with anti-ß-tubulin (A) and antiactin (B). Immunoprecipitates were processed for immunoblot analysis of s protein as described in Materials and Methods. Cytoskeletal proteins were detected by chemiluminescence as described in Materials and Methods. Numbers on the rightindicate the positions of molecular mass markers (kilodaltons). Blots are representative illustrations of results obtained in three independent experiments.

Immunofluorescent localization of _s

View larger version (106K): [in this window] [in a new window]   Figure 8. Effect of ACTH on immunofluorescence labeling of the s- and q-subunits of Gs and Gq proteins in rat glomerulosa cells. Rat glomerulosa cells were cultured for 3 days on plastic coverslips. After formaldehyde fixation, PIPES treatment, and permeabilization with 0.2% Triton X-100, cells were processed for immunofluorescence labeling using anti-s protein antibody and FITC for detection of s (A). Nonspecific labeling was obtained under similar conditions with a heat-inactivated anti-s antibody (B). Colocalization of q protein with microfilaments was revealed with a double stained preparation. Anti-q antibody was revealed by FITC labeling (C) and actin labeling with phalloidin/rhodamine (D) as described in Materials and Methods. All panels are shown at the same magnification of x3120. Images are representative illustration of more than 50 cells originating from 3 different experiments.

View larger version (138K): [in this window] [in a new window]   Figure 9. Electron microscopy of the Triton-insoluble preparation from adrenal glomerulosa cells. Electron microscopic examination of the Triton-insoluble preparation is shown at two magnifications. This preparation contained mainly microfilaments (MF) and spherical structures of variable sizes (arrow) and was free of other cellular structures. Magnification: A, x20,000; B, x66,000.

View larger version (18K): [in this window] [in a new window]   Figure 10. The effect of calcium on the dose-dependent effect of ACTH on cAMP production in human glomerulosa cells. Three-day cultures of human glomerulosa were labeled with [3H]adenine as described in Materials and Methods. Cells were incubated for 15 min in the absence (Control) or presence of increasing concentrations of ACTH. Experiments were performed in HBS buffer containing 1.8 mM CaCl2 (•), 0.4 mM CaCl2 (), or 100 nM CaCl2 (). The [3H]cAMP concentrations that accumulated were determined and expressed as a percentage of the total intracellular [3H]ATP calculated as described in Materials and Methods. Results are the mean ± SE of triplicate determinations in one experiment that is representative of three.

View larger version (80K): [in this window] [in a new window]   Figure 11. Effect of calcium-free medium on the distribution of actin in rat glomerulosa cells. Rat glomerulosa cells were cultured for 3 days on plastic coverslips and then incubated for 15 min (A and B) and 2 h (C) in the absence (A and C) or presence of 100 nM ACTH (B). After formaldehyde fixation and permeabilization with 0.1% Triton X-100, cells were processed for immunofluorescence labeling using rhodamine-phalloidin as described in Materials and Methods. All panels are shown at the same magnification of x3120. Images are representative illustrations of more than 50 cells originating from 2 different experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Cytoskeleton and cAMP production
Our results show that a 30-min incubation with colchicine completely blocks cAMP production induced by ACTH, whereas incubation with cytochalasin B decreases it by 50%. Moreover, our results indicate that colchicine decreases cAMP production stimulated by AlF^-₄ without hindering the effect of forskolin, indicating that microtubules are essential for G_s protein activation. These results confirm those of Feuilloley et al. (3) obtained using human adrenocortical cells, which demonstrate that colchicine affects ACTH-induced steroid secretion by acting before cAMP production. A close association between tubulin and G protein has been demonstrated by the studies of Rasenick et al. (16, 17, 30, 31, 32). Like G proteins, tubulin binds GTP, self-assembles, hydrolyzes GTP, and is ADP-ribosylated by cholera and pertussis toxin (30, 31). More importantly, Roychowdhury et al. (32) demonstrated that the transfer of GTP from tubulin to _s activates G_s. In addition, cytochalasin B inhibits stimulation induced by both AlF^-₄ and forskolin. These results are in agreement with the observations that both microtubules and microfilaments may be closely associated with the enzyme adenylyl cyclase (20) and its activation (21). The fact that colchicine blocks the fluoroaluminate response, but not the forskolin response, indicates that microtubules are involved upstream in the activation of adenylyl cyclase. This supports the observation made in frog and human adrenals, in which vinblastine had no effect on the stimulatory action of (Bu)₂cAMP on corticosteroidogenesis (3, 4). However, in Leydig cells (9), leukocytes (33), and lymphoma cells (34), colchicine increased both basal and hormone-stimulated cAMP production. Our results indicate that microtubules and microfilaments are essential for ACTH-stimulated cAMP production, but they act at different levels; microtubules are implicated in the activation of G_s protein, whereas microfilaments are essential for activation of both G_s and adenylyl cyclase.

ACTH and microfilament and microtubule organization
The rapid increase in membrane-associated actin during ACTH stimulation supports the importance of the role of microfilaments in the early events of ACTH action. The initial redistribution of actin at the membrane is followed by a net increase in actin polymerization, where stress fiber organization is intensified compared to that in control cells after a 2-h incubation with ACTH. Western blot analysis indicates that ACTH not only induces actin redistribution between 1–10 min of incubation, but also promotes polymerization after a 2-h incubation. The pattern of microfilament rearrangement after application of ACTH has been shown also by electron microscopic studies (1, 12, 35). The rapid increase in actin polymerization and association with the cell membrane has not been previously described in adrenal cells, but was observed in other cell types, such as blood cells (36, 37). Jennings et al. (36) demonstrated that a 15-sec stimulation of platelets with thrombin increases the amount of F-actin by 65% and increases the organization of actin filaments with other cytoskeletal proteins. In glomerulosa cells, our immunofluorescence studies show that during the same experimental period, the microtubular network is not significantly modified, although the amount of tubulin associated with the membrane did increase. A probable explanation for these discrepancies may be the alteration of the membrane during the process of permeabilization used to introduce the antitubulin antibody in the cell. This is obvious after comparison of Figs. 3 and 8D. In Fig. 3, the membrane actin network is more evident than in Fig. 8D, where double immunofluorescence was conducted. As reviewed recently (11), all of our data suggest that microtubules are associated with the early events of the action of ACTH rather than with the process of steroidogenesis (8, 38, 39).

Significance of _s-associated cytoskeleton during ACTH stimulation
Immunoblot analyses indicate that _s protein is not only present in the membrane, but is closely associated with microfilaments and microtubules. One-minute stimulation with ACTH strongly increases the amounts of _s and actin associated with the membrane, whereas the increased association of _s and tubulin with membrane is maintained for 15 min. We did not observe any changes in total tubulin or actin content under ACTH stimulation, indicating that ACTH promotes the polymerization of actin and tubulin either directly or indirectly without modification of the total tubulin and actin content.

Immunoblot experiments confirmed that _s protein is associated with both actin and tubulin, whereas immunofluorescence did not reveal overlapping labeling. The localization of _s as caveolae and the observations obtained from electron microscopy in which these caveolae are also present in Triton-insoluble preparations have been previously reported in MDCK kidneys cells (40). As G protein does not directly bind F-actin, it is likely that cytoskeletal association of G proteins may be mediated by some actin-associated proteins. Several newly identified proteins may be good candidates (41). In this respect, a recent publication reported the presence of a protein immunodetected around caveolae structures resistant to Triton X-100 extraction, which was subjected to dynamic movement under ACTH stimulation (42). This protein of 160 kDa could be an actin-binding protein, which may link G_s to microfilaments. Moreover, direct association of _s with actin and translocation from a low speed pellet (actin and associated proteins) to a high speed pellet (plasma membrane) have been documented during thrombin stimulation in platelets (43). All of these observations suggest that _s is associated with but not localized on cytoskeletal elements. Corroborating these results is the morphological evidence provided by Mattson and Kowal (12). These researchers observed that ACTH stimulation is accompanied by the formation of several small vesicles closely associated with microtubules. The observation that _s is associated with both microfilaments and microtubules is new in the adrenal. However, evidence for direct control of microfilament polymerization by G proteins has been described in neutrophils. Särndahl et al. (15) observed that G_n is associated with the cytoskeleton (primarily F-actin), but this association decreases or disappears under fluoroaluminate (AlF^-₄), GTPS, or fMet-Leu-Phe stimulation. Bengtsson et al. (44) also found that AlF^-₄ and GTPS are able to induce an increase in F-actin content, even when phospholipase C activity is inhibited.

On the other hand, the results presented in Fig. 10 show that cAMP production is impaired in a low or Ca²⁺-free medium despite ACTH binding to its receptor (45). This points out the crucial role of Ca²⁺ in the stimulating action of ACTH, although its exact target is not yet identified. Results presented in Fig. 11 show that a 15-min incubation in a Ca²⁺-free medium stabilizes actin fibers in a manner that cannot be activated by ACTH. A 2-h incubation in a Ca²⁺-free medium completely disrupts the microfilament network. Such observations have also been described by Castellino et al. (46). These researchers have shown that the caldesmon protein is responsible for this effect. The modification of actin fiber organization may be correlated with the absence of cAMP production under ACTH stimulation, supporting a role for microfilaments in the process of adenylyl cyclase activation.

In summary, the present study demonstrates that both microtubules and microfilaments are involved in the production of cAMP induced by ACTH in rat glomerulosa cells. Their role in the transduction signal of ACTH is due to their close association with _s protein. Our results support the concept 1) that microtubules may be implicated in the activation of G_s protein, whereas microfilaments are essential for both G_s and adenylyl cyclase activation; and 2) that rapid dynamic redistribution of both filaments from cytosol to membranes is responsible for cAMP production.

	Acknowledgments

 
The authors thank Lucie Chouinard and Liette Laflamme for experimental assistance, Dr. Jean-François Beaulieu for anti-IgG mouse rhodamine antibody for double staining experiments and fruitful advice and discussions, and Dr. Gilles Guillon (INSERM U-401, Montpellier, France) for the antibodies against G_q protein. We are greatly indebted to Dr. Bernard Schimmer for his critical review of the manuscript.

	Footnotes

 
¹ This work was supported by grants from the Medical Research Council of Canada and the Canadian Heart Foundation (to M.D.P. and N.G.-P.).

² Recipient of a Scholarship from Les Fonds de La Recherche en Santé du Québec.

Received June 24, 1996.

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